Recent developments in two-photon laser scanning microscopy (TPLSM) in combination with improvement of fluorescent calcium indicators have opened an unprecedented possibility for in vivo imaging of neuronal activity. Today, TPLSM of calcium signals is a widely used tool for studying of neuronal circuits and neuron-glial-vascular interactions in health and disease. Current techniques of introduction of calcium indicators into the brain tissue result in non-selective labeling of neurons and glia. While glial cells can be distinguished from neurons by adding selective markers, different types of neurons (excitatory and multiple types of inhibitory cells) cannot be discriminated. Yet, neuronal behavior is cell-type specific and understanding of the physiology and pathophysiology of identified neuronal cell types is of prime importance for development of new strategies of treatment and prevention of human disease. Thus, in vivo identification and functional imaging of specific cell types is a central unresolved problem in biomedical imaging. In principle, this can be achieved through use of genetic methods. However, cell-type specific transgenic expression has been only partially successful. The goal of this proposal is to develop an alternative strategy for identification of neuronal cell types taking an advantage of differential behavior of calcium signals in specific neuronal cell types. TPLSM will be used for detailed assessment of unique calcium "signatures" of cell-type specific neurophysiological properties observable in vivo in wild type animals. The findings will be validated by measurements from identified neurons in transgenic mice and in vitro brain slice preparations. The main deliverable of this project, a tool for "on-line" identification of excitatory and multiple types of inhibitory neurons, will enable simultaneous calcium imaging from multiple identified cell types with no need in additional promoter- specific genetic labeling. This approach will significantly advance in vivo two-photon calcium imaging used in basic neuroscience research and for investigation of animal models of human disease. PUBLIC HEALTH RELEVANCE Physiological identification of the neuronal cell types that comprise the mammalian brain is a central unresolved problem in neuroscience. The proposed project will advance application of two-photon laser scanning microscopy to imaging of neuronal assemblies by providing a tool for identification of specific cell types. Simultaneous in vivo imaging of identified neuronal phenotypes is crucial to define the normal state of physiology in neuronal networks and the degree of deviation in brain disease.